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Hepatitis Delta Virus Replication Affects the Expression of Host

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Hepatitis Delta Virus Replication Affects the Expression of Host HEPATITIS DELTA VIRUS REPLICATION AFFECTS THE EXPRESSION OF HOST GENES INVOLVED IN CELL CYCLE Gabrielle Goodrum THESIS SUBMITTED TO THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY Thesis Supervisor: Dr. Martin Pelchat Department of Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa © Gabrielle Goodrum, Ottawa, Canada, 2019 ABSTRACT The hepatitis delta virus (HDV) is the smallest human pathogenic RNA virus and relies heavily on host proteins for its replication. The objective of my research was to observe the effect of HDV replication on host gene expression, using a HEK-293-based cell system engineered to mimic HDV replication. A high-throughput sequencing was performed and allowed to establish a total of 3,561 genes differentially expressed by HDV RNA. Among those genes, 3,278 were upregulated by HDV RNA and 283 downregulated. A Gene Ontology (GO) enrichment analysis was performed on those dysregulated genes and revealed that upregulated genes were predominantly part of these four pathways: RNA processing, G- protein coupled receptor signaling pathway, protein transport, and organelle organization. On the other hand, downregulated genes were part of the nucleosome assembly pathway. The expression of several genes was confirmed by RT-qPCR. Moreover, protein complexes whose expression at the gene level was affected were identified. A total of 30 complexes were found to be significantly affected by HDV replication. Among them, we found many chromatin and histone related complexes. Lastly, a flow cytometry analysis revealed an increase in cell cycle arrest in G0/G1 and a reduction in the percentage of cell in S phase. Moreover, there was a difference in cell size for arrested cells in G0/G1 in HDV replicating cells. Overall, my results support the hypothesis that HDV replication induces cell cycle dysregulation. ii ACKNOWLEDGMENTS I would like to thank all the people who have supported and inspired me during my master. First, I would like to thank Dr. Martin Pelchat for this amazing opportunity, for his guidance and support along with this project. I thank him for the help he has given me with my bioinformatics analysis and for the confidence he had in me as and the freedom he gave me in this project. I would also like to say thank you to Lynda Rocheleau for carrying out many data processing steps. Thanks to Marc-André and Vera for their expert advice for the design of my flow cytometry experiments. I would also like to thank my thesis advisory committee Dr. Tommy Alain and Dr. Marc-André Langlois for their advice and support. I would like to thank Dorota Sikora, James Butcher and Patrick Taylor for being my mentors during my masters, for having the patience and the kindness to answer my questions and to have taught me so much. Also, thank you to all my friends from the Stintzi and the Fullerton laboratory for being there when I needed it. I would also like to thank Kevin who was my moral support during all my studies, which encouraged me and believed in me. For his patience to listen to my oral presentations, again and again. Thank you for teaching me so many informatics basic concepts. Finally, I would like to thank my parents for their constant support during my studies, to push me to surpass myself and for believing in me. I am also grateful for the McGill University and Génome Québec Innovation Center, as well as Dr. John Taylor (Fox Chase Cancer Center, USA) for kindly providing the cell lines. This study was supported by a NSERC discovery grant. iii TABLE OF CONTENT Table of Contents ABSTRACT ....................................................................................................................................................... II ACKNOWLEDGMENTS ................................................................................................................................ III LIST OF ACRONYMS .................................................................................................................................... VI LIST OF FIGURES ....................................................................................................................................... VIII LIST OF TABLES ............................................................................................................................................ IX CHAPTER 1: INTRODUCTION ...................................................................................................................... 1 1.1 DISCOVERY AND CLASSIFICATION ................................................................................................................ 2 1.2 EPIDEMIOLOGY ............................................................................................................................................ 4 1.3 PATHOGENICITY ........................................................................................................................................... 5 1.4 VIRION AND GENOME STRUCTURE ................................................................................................................ 6 1.5 SYMMETRICAL ROLLING CIRCLE REPLICATION ........................................................................................... 13 1.6 DELTA ANTIGENS ........................................................................................................................................ 17 1.6.1 Function of HDAg-S .......................................................................................................................... 20 1.6.2 Function of HDAg-L .......................................................................................................................... 21 1.7 POST-TRANSLATIONAL MODIFICATIONS OF HDAGS ................................................................................... 23 1.8 INTERACTION OF HDV WITH HOST CELLULAR PROTEINS .......................................................................... 25 1.8.1 HDAg interactions with host proteins ................................................................................................ 25 1.8.2 HDV RNA interaction with host proteins ........................................................................................... 27 1.9 MODELS DEVELOPED TO STUDY HDV REPLICATION .................................................................................. 32 1.10 HOW HDV AFFECTS ITS HOST CELL.......................................................................................................... 34 1.10.1 Proteomic analyses .......................................................................................................................... 34 1.10.2 Interferon response .......................................................................................................................... 37 1.10.2 Stress and foci formation ................................................................................................................. 40 1.11 RATIONALE, HYPOTHESIS, AND OBJECTIVES ............................................................................................. 42 CHAPTER 2: MATERIALS AND METHODS ............................................................................................. 43 CELL CULTURE AND INDUCTION....................................................................................................................... 44 RNA ISOLATION, LIBRARY CONSTRUCTION AND ILLUMINA DEEP-SEQUENCING .............................................. 44 DIFFERENTIAL EXPRESSION ANALYSIS OF TRANSCRIPT AND DATA PROCESSING............................................... 45 GENE ONTOLOGY ENRICHMENT ....................................................................................................................... 46 IDENTIFICATION OF AFFECTED COMPLEXES...................................................................................................... 46 REVERSE TRANSCRIPTION AND QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ................................ 47 WESTERN BLOT ASSAY .................................................................................................................................... 48 FLOW CYTOMETRY ANALYSIS .......................................................................................................................... 49 Sample preparation ..................................................................................................................................... 49 Data processing: gating and rescaling ....................................................................................................... 50 Data analysis .............................................................................................................................................. 51 CHAPTER 3: RESULTS .................................................................................................................................. 53 3.1 TRANSCRIPTOME ANALYSIS OF GENES AFFECTED BY HDV ........................................................................ 54 3.1.1 Sample preparation and data processing .......................................................................................... 54 3.1.2. Gene expression analysis .................................................................................................................. 63 3.2 ENRICHMENT OF GENE ONTOLOGY TERM AND BIOLOGICAL PROCESSES AFFECTED BY HDV REPLICATION 71 3.3 WEIGHTED CORRELATION
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